SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 enables scarless genome editing.
• Existing fission yeast CRISPR systems require complex cloning and can cause Cas9 toxicity.
• Overexpression of Cas9 from the strong adh1 promoter is toxic to S. pombe.

Purpose of the Study:

• To develop an improved, user-friendly CRISPR/Cas9 system for fission yeast (Schizosaccharomyces pombe).
• To overcome limitations of existing systems, including tedious cloning and Cas9 toxicity.
• To enhance the efficiency and versatility of genome editing in S. pombe.

Main Methods:

• Developed the SpEDIT system with a codon-optimized Cas9 sequence for S. pombe.
• Utilized a modular design with sgRNA fused to a hepatitis delta virus (HDV) ribozyme for expression.
• Employed Golden Gate assembly for one-step replacement of GFP with synthesized sgRNAs.
• Expressed sgRNA cassettes using RNA polymerase III from tRNA gene sequences.

Main Results:

• Achieved 100% mutagenesis efficiency for targeted point mutations in ade6+ or ura4+ genes.
• Enabled insertion, tagging, and deletion events with minimal effort.
• Facilitated simultaneous editing of two independent non-homologous loci.
• Demonstrated reduced toxicity compared to existing S. pombe editing systems.

Conclusions:

• SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure for fission yeast.
• The system significantly improves the genome editing toolbox for S. pombe.
• SpEDIT overcomes limitations of previous systems, offering enhanced efficiency and reduced toxicity.